Protein disulfide isomerase and ABC transporter homologous proteins involved in the regulation of energy homeostasis

ABSTRACT

The present invention discloses three novel proteins regulating the energy homeostasis and the metabolism of triglycerides, and polynucleotides, which identify and encode the proteins disclosed in this invention. The invention also relates to vectors, host cells, antibodies, and recombinant methods for producing the polypeptides and polynucleotides of the invention. The invention also relates to the use of these sequences in the diagnosis, study, prevention, and treatment of diseases and disorders related to body-weight regulation, for example, but not limited to, metabolic diseases such as obesity, as well as related disorders such as adipositas, eating disorders, wasting syndromes (cachexia), pancreatic dysfunctions (such as diabetes mellitus), hypertension, pancreatic dysfunctions, arteriosclerosis, coronary artery disease (CAD), coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, cancer, e.g. cancers of the reproductive organs, sleep apnea, and others.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT Patent Application No.PCT/EP02/03540 filed Mar. 28, 2002 and European Patent Applications No.EP01 108 315.1 filed Apr. 2, 2001 and EP01 113 419.4 filed Jun. 1, 2001.

DESCRIPTION

This invention relates to the use of nucleic acid and amino acidsequences of protein disulfide isomerase and ABC transporter homologousproteins, and to the use of these sequences in the diagnosis, study,prevention, and treatment of diseases and disorders related tobody-weight regulation, for example, but not limited to, metabolicdiseases such as obesity, as well as related disorders such asadipositas, eating disorders, wasting syndromes (cachexia), pancreaticdysfunctions (such as diabetes mellitus), hypertension,arteriosclerosis, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancer, e.g. cancers of thereproductive organs, sleep apnea, and others.

Obesity is one of the most prevalent metabolic disorders in the world.It is still poorly understood human disease that becomes more and morerelevant for western society. Obesity is defined as an excess of bodyfat, frequently resulting in a significant impairment of health. Besidessevere risks of illness such as diabetes, hypertension and heartdisease, individuals suffering from obesity are often isolated socially.Human obesity is strongly influenced by environmental and geneticfactors, whereby the environmental influence is often a hurdle for theidentification of (human) obesity genes. Obesity is influenced bygenetic, metabolic, biochemical, psychological, and behavioral factors.As such, it is a complex disorder that must be addressed on severalfronts to achieve lasting positive clinical outcome. Obese individualsare prone to ailments including: diabetes mellitus, hypertension,coronary heart disease, hypercholesterolemia, dyslipidemia,osteoarthritis, gallstones, cancer, e.g. cancers of the reproductiveorgans, and sleep apnea.

Obesity is not to be considered as a single disorder but a heterogeneousgroup of conditions with (potential) multiple causes. Obesity is alsocharacterized by elevated fasting plasma insulin and an exaggeratedinsulin response to oral glucose intake (Koltermann, J. Clin. Invest 65,1980, 1272–1284) and a clear involvement of obesity in type 2 diabetesmellitus can be confirmed (Kopelman, Nature 404, 2000, 635–643).

Even if several candidate genes have been described which are supposedto influence the homeostatic system(s) that regulate body mass/weight,like leptin, VCPI, VCPL, or the peroxisome proliferator-activatedreceptor-gamma co-activator, the distinct molecular mechanisms and/ormolecules influencing obesity or body weight/body mass regulations arenot known.

Therefore, the technical problem underlying the present invention was toprovide for means and methods for modulating (pathological) metabolicconditions influencing body-weight regulation and/or energy homeostaticcircuits. The solution to said technical problem is achieved byproviding the embodiments characterized in the claims.

Accordingly, the present invention relates to genes with novel functionsin body-weight regulation, energy homeostasis, metabolism, and obesity.The present invention discloses specific genes involved in theregulation of body-weight, energy homeostasis, metabolism, and obesity,and thus in disorders related thereto such as eating disorder, cachexia,diabetes mellitus, hypertension, coronary heart disease,hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, cancer,e.g. cancers of the reproductive organs, and sleep apnea. The presentinvention describes human protein disulfide isomerase, MRP4, and ABC8(white) genes as being involved in those conditions mentioned above.

Protein disulfide isomerase (PDI) is a protein-thiol oxidoreductase thatcatalyzes the folding of protein disulfides. PDI has been demonstratedto participate in the regulation of reception function, cell-cellinteraction, gene expression, and actin filament polymerization. PDI hasacts as a chaperone and subunit of prolyl 4-hydroxylase and microsomaltriglyceride transfer protein (MTP). MTP is accelerating the transportof triglyceride, cholesteryl ester, and phospholipid between membranes(Berriot-Varoqueaux et al., 2000, Annu Rev Nutr. 20, 663–697). Mutationsin MTP are the cause for abetalipoproteinemia, a hereditary disease withlimited production of chylomicrons and very low-density lipoproteins(VLDL) in the intestine and liver (Rehberg et al., 1996, J Biol Chem.271(47), 29945–52). Intracellular VLDL is associated with chaperonesincluding PDI and glucose regulated protein 94 (GRP94, endoplasmin) andassembles with apolipoprotein B (Berriot-Varoqueaux et al., SUPRA).These chaperones are endogenous substrates of sphingosine-dependentkinases (SDKs) and regulated by signal transduction pathways (see, forexample, Megidish et al., 1999, Biochemistry. 38(11), 3369–78).

The chaperones are found in the endoplasmic reticulum where thelipidation of lipoproteins like apolipoprotein B might take place(Hussain et al., 1997, Biochemistry. 36(42), 13060–7.; Wu. et al., 1996,J Biol Chem. 271(17), 10227–81). In addition, the secretion ofapolipoprotein B is dependent on PDI (Wang et al., 1997, J. Biol. Chem.272(44), 27644–51).

The formation or assembly of additional proteins strongly depends on theactivity of specific groups of chaperones. PDI is regulating theformation of native insulin from its precursors and the insulindegradation (Tang et al., 1988, Biochem J. 255(2), 451–5; Duckworth etal., 1998, Endocr Rev. 19(5), 608–24). Insulin signaling is crucial forthe proper regulation of blood glucose levels and lipid metabolism.

Dietary energy tissue-specifically regulates endoplasmic reticulumchaperone gene expression in the liver of mice, especially glucoseregulated proteins (Dhahbi et al., 1997, J Nutr. 127(9), 1758–64).

Additionally PDI mRNA is strongly expressed in adipose tissue (Klaus etal., 1990, Mol Cell Endocrinol. 73(2–3), 105–10), emphasizing theirimportant roles in metabolic pathways.

Chaperones are also essential for the cellular protection against stressin its different forms like oxidative, heat shock or hypoglycemic stress(Barnes & Smoak, 2000, Anat Embryol. 202(1), 67–74, Lee, 1992. Curr OpinBiol. 4(2), 267–73) preventing cells to undergo apoptosis.

Furthermore, chaperones are involved in different diseases likeAlzheimer's Disease (Yoo et al., 2001, Biochem Biophys Res Commun.280(1), 249–58), Parkinson (Duan & Mattson, 1999, J Neurosci Res.59(13), 195–206), Rheumatoid Arthritis (Corrigall et al., 2001, JImmunol. 166(3), 1492–98) or neuropsychological disease leading tosuicide of patients (Bown et al., 2000, Neuropsychopharmacology. 22(3),327–32).

ATP-binding cassette (ABC) genes encode a family of transport proteinsthat are known to be involved in a number of human genetic diseases. Forexample, polymorphisms of the human homologue of the Drosophila whitegene are associated with mood and panic disorders (Nakamura et al. 1999Mol Psychiatry. 4(2):155–62). Mutations in the canilicular multispecificorganic anion transporter (cMOAT) gene could be the reason for theDubin-Johnson syndrome leading to hyperbilirubinemia II (Wada et al.1998, Hum Mol Genet. 7(2):203–7). The rod photoreceptor-specific ABCtransporter (ABCR) is responsible for the Stargardt disease (Allikmetset al. 1997, Nat Genet. 15(3):236–46). The gene encoding ATP-bindingcassette transporter 1 (ABC1) is mutated in Tangier disease leading tothe absence of plasma high-density lipoprotein (HDL) and deposition ofcholesteryl esters in the reticulo-endothelial system with splenomegalyand enlargement of tonsils and lymph nodes (Brooks-Wilson et al. 1999Nat Genet. 22(4):336–45, Bodzioch et al. 1999 Nat Genet. 22(4):347–51,Rust et al. 1999, Nat Genet. 22(4):352–5). Furthermore a subgroup of ABCtransporters are involved in cellular detoxification causing multidrugresistance that counteracts e.g. anti-cancer or HIV treatment (Schuetzet al. 1999 Nat Med. 1999 (9):1048–51).

ABC transporters transport several classes of molecules. ABC1 (ABCA1),the gene mutated in Tangier disease, mediates apoAI associated export ofcholesterol and phospholipids from the cell and is regulated bycholesterol efflux ((Brooks-Wilson et al. 1999 Nat Genet. 22(4):336–45,Bodzioch et al. 1999 Nat Genet. 22(4):347–51, Rust et al. 1999, NatGenet. 22(4):352–5Brooks-Wilson et al. 1999, Bodzioch et al. 1999, Rustet al. 1999). ABC1 is expressed on the plasma membrane and Golgi complexand the lipid export process needs vesicular budding between Golgi andplasma membrane that is disturbed in Tangier disease. LDL and HDL₃regulate the expression of ABC1 in macrophages (Orsó et al. 2000 NatGenet. 24:192–6).

The expression of the human homologue of the Drosophila white gene (ABC8or ABCG1) is induced in monocyte-derived macrophages during cholesterolinflux mediated by LDL and is downregulated through lipid effluxmediated by cholesterol acceptor HDL₃. ABC8 is expressed on the cellsurface and intracellular compartments of cholesterol-loaded macrophagesand its expression is also regulated by oxysterols that act throughnuclear oxysterol receptors, liver X receptors (LXRs) and by retinoid Xreceptor ligand. Therefore, ABC8 activity in macrophages might becrucial for cholesterol metabolism and the development ofarteriosclerosis similar to ABC1. LXR expression is regulated throughPPARg gamma signalling that is essential for adipogenesis and thereforePPARg gamma signalling might regulate the expression of ABC1 and ABC8transporters at least in macrophages (Klucken et al. 2000 Proc Natl AcadSci USA. 97(2):817–22., Venkateswaran et al. 2000 J Biol Chem.275(19):14700–7).

Another subgroup of ABC transporters mediates cellular detoxificationand is therefore named Multidrug Resistance-associated Proteins (MRPs)or canilicular Multispecific Organic Anion Transporters (cMOATs). MRP1has high activity towards compounds conjugated to glutathione (GSH),glucuronide or sulfate and transports sphingolipids, eicosanoids,phosphatidylcholine and phosphatidylethanolamine analogues but the mainfunction is the cellular detoxification. MRP4 (MOAT-B) overexpression isassociated with high-level resistance to the nucleoside analogues9-(2-phosphonylmethoxyethyl)adenine and azidothymidine monophosphate,both of which are used as anti-human immunodeficiency virus (HIV) drugs(Schuetz et al. 1999 Nat Med. 5(9):1048–51). MRP4 function in lipidtransport is unknown despite the fact that LDL and HDL₃ regulate itsexpression in macrophages.

So far, it has not been described that protein disulfide isomerase orABC transporters and the homologous human proteins are involved in theregulation of energy homeostasis and body-weight regulation and relateddisorders, and thus, no functions in metabolic diseases and otherdiseases as listed above have been discussed. In this invention wedemonstrate that the correct gene doses of protein disulphide isomeraseand/or of two ABC transporter genes are essential for maintenance ofenergy homeostasis. A genetic screen was used to identify that theprotein disulfide isomerase gene, the MRP4 gene, and/or the white (ABC8)gene cause obesity in Drosophila melanogaster, reflected by asignificant increase of triglyceride content, the major energy storagesubstance.

Polynucleotides encoding proteins with homologies to protein disulfideisomerase or ABC transporters present the opportunity to investigatediseases and disorders, including metabolic diseases and disorders suchas obesity, as well as related disorders such as described above.Molecules related to protein disulfide isomerase and ABC transporterssatisfy a need in the art by providing new compositions useful indiagnosis, treatment, and prognosis of diseases and disorders, includingmetabolic diseases and disorders such as described above.

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meanings as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure.

The present invention discloses a novel protein disulfide isomerasehomologous protein and two novel ABC transporter homologous proteinsregulating the energy homeostasis and the fat metabolism, especially themetabolism and storage of triglycerides, and polynucleotides, oftriglycerides, and polynucleotides, which identify and encode theproteins disclosed in this invention. The invention also relates tovectors, host cells, antibodies, and recombinant methods for producingthe polypeptides and polynucleotides of the invention. The inventionalso relates to the use of these sequences in the diagnosis, study,prevention, and treatment of diseases and disorders related tobody-weight regulation, for example, but not limited to, metabolicdiseases such as obesity, as well as related disorders such asadipositas, eating disorders, wasting syndromes (cachexia), pancreaticdysfunctions (such as diabetes mellitus), hypertension,arteriosclerosis, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancer, e.g. cancers of thereproductive organs, sleep apnea, and others.

Protein disulfide isomerase and ABC transporter homologous proteins andnucleic acid molecules coding therefor are obtainable from insect orvertebrate species, e.g. mammals or birds. Particularly preferred arehuman protein disulfide isomerase and ABC transporter homologous nucleicacids, particularly nucleic acids encoding human protein disulfideisomerase homologous protein (MGC3178; Genbank Accession No.NM_(—)030810 (identical to Genbank Accession No. BC001199) for the mRNA;NP_(—)110437.1 (identical to Genbank Accession No. AAH01199) for theprotein), human ATP-binding cassette, subfamily C(CFTR/MRP), member 4(ABCC4; MRP4; Genbank Accession No. NM_(—)005845 for the mRNA;NP_(—)005836.1 for the protein), and human ATP-binding cassette,sub-family G (WHITE), member 1 (ABCG1; WHITE; Genbank Accession No.XM_(—)009777 for the mRNA; XP_(—)009777.3 for the protein). Alsoparticularly preferred are Drosophila protein disulfide isomerasehomologous and ABC transporter homologous nucleic acids and polypeptidesencoded thereby. In a preferred embodiment the present invention alsocomprises so-called “ABC-tran” domains of the proteins and nucleic acidmolecules coding therefor.

The invention particularly relates to a nucleic acid molecule encoding apolypeptide contributing to regulating the energy homeostasis and themetabolism of triglycerides, wherein said nucleic acid moleculecomprises

-   (a) the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or    SEQ ID NO:5,-   (b) a nucleotide sequence which hybridizes at 66° C. in a solution    containing 0.2×SSC and 0.1% SDS to the complementary strand of a    nucleic acid molecule encoding the amino acid sequence of SEQ ID    NO:2, SEQ ID NO:4, or SEQ ID NO:6,-   (c) a sequence corresponding to the sequences of (a) or (b) within    the degeneration of the genetic code,-   (d) a sequence which encodes a polypeptide which is at least 85%,    preferably at least 90%, more preferably at least 95%, more    preferably at least 98% and up to 99,6% identical to SEQ ID NO:2,    SEQ ID NO:4, or SEQ ID NO:6,-   (e) a sequence which differs from the nucleic acid molecule of (a)    to (d) by mutation and wherein said mutation causes an alteration,    deletion, duplication or premature stop in the encoded polypeptide    or-   (f) a partial sequence of any of the nucleotide sequences of (a)    to (e) having a length of at least 15 bases, preferably at least 20    bases, more preferably at least 25 bases and most preferably at    least 50 bases.

The invention is based on the discovery that protein disulfide isomerasehomologous proteins (particularly PDI-like; referred to as DevG20) andABC transporter homologous proteins (particularly MRP4 and WHITE; hereinreferred to as DevG4 and DevG22, respectively) and the polynucleotidesencoding these, are involved in the regulation of triglyceride storageand therefore energy homeostasis. The invention describes the use ofthese compositions for the diagnosis, study, prevention, or treatment ofdiseases and disorders related to such cells, including metabolicdiseases such as obesity, as well as related disorders such asadipositas, eating disorders, wasting syndromes (cachexia), pancreaticdysfunctions (such as diabetes mellitus), hypertension,arteriosclerosis, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancer, e.g. cancers of thereproductive organs, sleep apnea, and others.

To find genes with novel functions in energy homeostasis, metabolism,and obesity, a functional genetic screen was performed with the modelorganism Drosophila melanogaster (Meigen). One resource for screeningwas a proprietary Drosophila melanogaster stock collection of EP-lines.The P-vector of this collection has Gal4-UAS-binding sites fused to abasal promoter that can transcribe adjacent genomic Drosophila sequencesupon binding of Gal4 to UAS-sites. This enables the EP-line collectionfor overexpression of endogenous flanking gene sequences. In addition,without activation of the UAS-sites, integration of the EP-element intothe gene is likely to cause a reduction of gene activity, and allowsdetermining its function by evaluating the loss-of-function phenotype.

Triglycerides are the most efficient storage for energy in cells. Inorder to isolate genes with a function in energy homeostasis, severalthousand proprietary EP-lines were tested for their triglyceride contentafter a prolonged feeding period (see Examples for more detail). Lineswith significantly changed triglyceride content were selected aspositive candidates for further analysis.

Obese people mainly show a significant increase in the content oftriglycerides. In this invention, the content of triglycerides of a poolof flies with the same genotype after feeding for six days was analyzedusing a triglyceride assay, as, for example, but not for limiting thescope of the invention, is described below in the EXAMPLES section.

The invention encompasses polynucleotides that encode DevG20, DevG4,DevG22, and homologous proteins. Accordingly, any nucleic acid sequence,which encodes the amino acid sequences of DevG20, DevG4, or DevG22, canbe used to generate recombinant molecules that express DevG20, DevG4, orDevG22. In a particular embodiment, the invention encompasses thenucleic acid sequence of 1693 base pairs (PDI, referred to as DevG20 SEQID NO:1) as shown in FIG. 4A, the nucleic acid sequence of 4487 basepairs (MRP4, referred to as DevG4, SEQ ID NO:3) as shown in FIG. 9A, andthe nucleic acid sequence of 2459 base pairs (ABC8, or white, referredto as DevG22 SEQ ID NO:5) as shown in FIG. 15A. It will be appreciatedby those skilled in the art that as a result of the degeneracy of thegenetic code, a multitude of nucleotide sequences encoding DevG20,DevG4, or DevG22, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequences of naturally occurring DevG20, DevG4, or DevG22, and all suchvariations are to be considered as being specifically disclosed.Although nucleotide sequences which encode DevG20, DevG4, or DevG22 andvariants thereof are preferably capable of hybridising to the nucleotidesequences of the naturally occurring DevG20, DevG4, or DevG22 underappropriately selected conditions of stringency, it may be advantageousto produce nucleotide sequences encoding DevG20, DevG4, or DevG22 orderivatives thereof possessing a substantially different codon usage.Codons may be selected to increase the rate at which expression of thepeptide occurs in a particular prokaryotic or eukaryotic host inaccordance with the frequency with which particular codons are utilisedby the host. Other reasons for substantially altering the nucleotidesequence encoding DevG20, DevG4, or DevG22 and derivatives thereofwithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequences. The invention also encompasses production of DNA sequences,or portions thereof, which encode DevG20, DevG4, or DevG22 andderivatives thereof, entirely by synthetic chemistry. After production,the synthetic sequence may be inserted into any of the many availableexpression vectors and cell systems using reagents that are well knownin the art at the time of the filing of this application. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding DevG20, DevG4, or DevG22 or any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridising to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:1, SEQ ID NO:3, and in SEQ ID NO:5,under various conditions of stringency. Hybridisation conditions arebased on the melting temperature (Tm) of the nucleic acid bindingcomplex or probe, as taught in Wahl, G. M. and S. L. Berger (1987:Methods Enzymol. 152:399–407) and Kimmel, A. R. (1987; Methods Enzymol.152:507–511), and may be used at a defined stringency. Preferably,hybridization under stringent conditions means that after washing for 1h with 1×SSC and 0.1% SDS at 50° C., preferably at 55° C., morepreferably at 62° C. and most preferably at 68° C., particularly for 1 hin 0.2×SSC and 0.1% SDS at 50° C., preferably at 55° C., more preferablyat 62° C. and most preferably at 68° C., a positive hybridization signalis observed. Altered nucleic acid sequences encoding DevG20, DevG4, orDevG22 which are encompassed by the invention include deletions,insertions, or substitutions of different nucleotides resulting in apolynucleotide that encodes the same or a functionally equivalentprotein.

The encoded proteins may also contain deletions, insertions, orsubstitutions of amino acid residues, which produce a silent change andresult in a functionally equivalent protein. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the biological activity ofDevG20, DevG4, or DevG22 is retained. For example, negatively chargedamino acids may include aspartic acid and glutamic acid; positivelycharged amino acids may include lysine and arginine; and amino acidswith uncharged polar head groups having similar hydrophilicity valuesmay include leucine, isoleucine, and valine; glycine and alanine;asparagine and glutamine; serine and threonine; phenylalanine andtyrosine.

Also included within the scope of the present invention are alleles ofthe genes encoding DevG20, DevG4, or DevG22. As used herein, an “allele”or “allelic sequence” is an alternative form of the gene, which mayresult from at least one mutation in the nucleic acid sequence. Allelesmay result in altered mRNAs or polypeptides whose structures or functionmay or may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes, which give rise to alleles,are generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any embodiments of the invention. Themethods may employ such enzymes as the Klenow fragment of DNA polymeraseI, SEQUENASE DNA Polymerase (US Biochemical Corp, Cleveland Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproof-reading exonucleases such as the ELONGASE Amplification System(GIBCO/BRL, Gaithersburg, Md.). Preferably, the process is automatedwith machines such as the Hamilton MICROLAB 2200 (Hamilton, Reno Nev.),Peltier thermal cycler (PTC200; MJ Research, Watertown, Mass.) and theABI 377 DNA sequencers (Perkin Elmer). The nucleic acid sequencesencoding DevG20, DevG4, or DevG22 may be extended utilising a partialnucleotide sequence and employing various methods known in the art todetect upstream sequences such as promoters and regulatory elements. Forexample, one method which may be employed, “restriction-site” PCR, usesuniversal primers to retrieve unknown sequence adjacent to a known locus(Sarkar, G. (1993) PCR Methods Applic. 2:318–322). In particular,genomic DNA is first amplified in the presence of primer to linkersequence and a primer specific to the known region. The amplifiedsequences are then subjected to a second round of PCR with the samelinker primer and another specific primer internal to the first one.Products of each round of PCR are transcribed with an appropriate RNApolymerase and sequenced using reverse transcriptase. Inverse PCR mayalso be used to amplify or extend sequences using divergent primersbased on a known region (Triglia, T. et al. (1988) Nucleic Acids Res.16:8186). The primers may be designed using OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to 22–30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequencetemperatures about 68–72° C. The method uses several restriction enzymesto generate suitable fragments. The fragment is then circularised byintramolecular ligation and used as a PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (PCR MethodsApplic. 1:111–119). In this method, multiple restriction enzymedigestions and ligations also are used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055–3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences, which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions. Capillary electrophoresis systems,which are commercially available, may be used to analyse the size orconfirm the nucleotide sequence of sequencing or PCR products. Inparticular, capillary sequencing may employ flowable polymers forelectrophoretic separation, four different fluorescent dyes (one foreach nucleotide) which are laser activated, and detection of the emittedwavelengths by a charge coupled devise camera. Output/light intensitymay be converted to electrical signal using appropriate software (e.g.GENOTYPER and SEQUENCE NAVIGATOR, Perkin Elmer) and the entire processfrom loading of samples to computer analysis and electronic data displaymay be computer controlled. Capillary electrophoresis is especiallypreferable for the sequencing of small pieces of DNA, which might bepresent in limited amounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences whichencode DevG20, DevG4, or DevG22 or fragments thereof, or fusion proteinsor functional equivalents thereof, may be used in recombinant DNAmolecules to direct expression of DevG20, DevG4, or DevG22 inappropriate host cells. Due to the inherent degeneracy of the geneticcode, other DNA sequences, which encode substantially the same, or afunctionally equivalent amino acid sequence may be produced and thesesequences may be used to clone and express DevG20, DevG4, or DevG22. Aswill be understood by those of skill in the art, it may be advantageousto produce DevG20-encoding, DevG4-encoding, and DevG22-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence. The nucleotide sequences of the presentinvention can be engineered using methods generally known in the art inorder to alter DevG20, DevG4, or DevG22 encoding sequences for a varietyof reasons, including but not limited to, alterations which modify thecloning, processing, and/or expression of the gene product. DNAshuffling by random fragmentation and PCR reassembly of gene fragmentsand synthetic oligonucleotides may be used to engineer the nucleotidesequences. For example, site-directed mutagenesis may be used to insertnew restriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding DevG20, DevG4, or DevG22 maybe ligated to a heterologous sequence to encode a fusion protein. Forexample, to screen peptide libraries for inhibitors of DevG20, DevG4, orDevG22 activities, it may be useful to produce chimeric proteins thatcan be recognised by a commercially available antibodies. A fusionprotein may also be engineered to contain a cleavage site locatedbetween the DevG20, DevG4, or DevG22 encoding sequence and theheterologous protein sequences, so that DevG20, DevG4, or DevG22 may becleaved and purified away from the heterologous moiety. In anotherembodiment, sequences encoding DevG20, DevG4, or DevG22 may besynthesised, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.7:215–223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.7:225–232). Alternatively, the proteins themselves may be produced usingchemical methods by synthesising the amino acid sequence, or a portionthereof. For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202–204) and automated synthesis may be achieved, for example, usingthe ABI 431A peptide synthesiser (Perkin Elmer). The newly synthesisedpeptide may be substantially purified by preparative high performanceliquid chromatography (e.g., Creighton, T. (1983) Proteins, Structuresand Molecular Principles, WH Freeman and Co., New York, N.Y.). Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure;Creighton, supra). Additionally, the amino acid sequences, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

In order to express a biologically active DevG20, DevG4, or DevG22, thenucleotide sequences coding therefor or functional equivalents, may beinserted into appropriate expression vectors, i.e., a vector, whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods, which are well known to thoseskilled in the art, may be used to construct expression vectors andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques. synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilised to containand express sequences encoding DevG20, DevG4, or DevG22. These include,but are not limited to, micro-organisms such as bacteria transformedwith recombinant bacteriophage, plasmid, or cosmid DNA expressionvectors; yeast transformed with yeast expression vectors; insect cellsystems infected with virus expression vectors (e.g., baculovirus);plant cell systems transformed with virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or withbacterial expression vectors (e.g., Ti or PBR322 plasmids); or animalcell systems. The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector-enhancers, promoters, 5′ and 3′untranslated regions which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilised, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters and enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters and leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequences, vectors basedon SV40 or EBV may be used with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the protein. For example, when largequantities of protein are needed for the induction of antibodies,vectors, which direct high level expression of fusion proteins that arereadily purified, may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asthe BLUESCRIPT phagemid (Stratagene), in which the sequence encodingDevG20, DevG4, or DevG22 may be ligated into the vector in frame withsequences for the amino-terminal Met and the subsequent 7 residues ofβ-galactosidase so that a hybrid protein is produced; pIN vectors (VanHeeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503–5509); andthe like. PGEX vectors (Promega, Madison, Wis.) may also be used toexpress foreign polypeptides as fusion proteins with GlutathioneS-Transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems may be designed to includeheparin, thrombin, or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill. In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.,(supra) and Grantet al. (1987) Methods Enzymol. 153:516–544.

In cases where plant expression vectors are used, the expression ofsequences encoding DevG20, DevG4, or DevG22 may be driven by any of anumber of promoters. For example, viral promoters such as the 35S and19S promoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307–311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671–1680; Broglie, R. et al. (1984) Science 224:838–843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85–105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191–196).

An insect system may also be used to express DevG20, DevG4, or DevG22.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and place under control of the polyhedrin promoter.Successful insertions will render the polyhedrin gene inactive andproduce recombinant virus lacking coat protein. The recombinant virusesmay then be used to infect, for example, S. frugiperda cells ofTrichoplusia larvae in which DevG20, DevG4, or DevG22 may be expressed(Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. 91:3224–3227).

In mammalian host cells, a number of viral-based expression systems maybe utilised. In cases where an adenovirus is used as an expressionvector, sequences encoding DevG20, DevG4, or DevG22 may be ligated intoan adenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain viable viruseswhich are capable of expression in infected host cells (Logan, J. andShenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655–3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation. Such signals include the ATG initiation codon and adjacentsequences. In cases where sequences encoding DevG20, DevG4, or DevG22,initiation codons, and upstream sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a portion thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125–162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressDevG20, DevG4, or DevG22 may be transformed using expression vectorswhich may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1–2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells, which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type. Any number ofselection systems may be used to recover transformed cell lines. Theseinclude, but are not limited to, the herpes simplex virus thymidinekinase (Wigler, M. et al. (1977) Cell 11:223–32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817–23) genes,which can be employed in tk- or aprt-, cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567–70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1–14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilise indole in place of tryptophan, or hisD,which allows cells to utilise histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047–51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β-glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121–131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequences encoding DevG20,DevG4, or DevG22 are inserted within a marker gene sequence, recombinantcells containing sequences encoding DevG20, DevG4, or DevG22 can beidentified by the absence of marker gene function. Alternatively, amarker gene can be placed in tandem with sequences encoding DevG20,DevG4, or DevG22 under the control of a single promoter. Expression ofthe marker gene in response to induction or selection usually indicatesexpression of the tandem gene as well. Alternatively, host cells, whichcontain and express the nucleic acid sequences encoding DevG20, DevG4,or DevG22, may be identified by a variety of procedures known to thoseof skill in the art. These procedures include, but are not limited to,DNA-DNA, or DNA-RNA hybridisation and protein bioassay or immunoassaytechniques which include membrane, solution, or chip based technologiesfor the detection and/or quantification of nucleic acid or protein.

The presence of polynucleotide sequences encoding DevG20, DevG4, orDevG22 can be detected by DNA-DNA or DNA-RNA hybridisation oramplification using probes or portions or fragments of polynucleotidesspecific for DevG20, DevG4, or DevG22. Nucleic acid amplification basedassays involve the use of oligonucleotides or oligomers based on thesequences specific for DevG20, DevG4, or DevG22 to detect transformantscontaining DNA or RNA encoding DevG20, DevG4, or DevG22. As used herein“oligonucleotides” or “oligomers” refer to a nucleic acid sequence of atleast about 10 nucleotides and as many as about 60 nucleotides,preferably about 15 to 30 nucleotides, and more preferably about 20–25nucleotides, which can be used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofDevG20, DevG4, or DevG22, using either polyclonal or monoclonalantibodies specific for the protein are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilising monoclonal antibodies reactive totwo non-interfering epitopes on the protein is preferred, but acompetitive binding assay may be employed. These and other assays aredescribed, among other places, in Hampton, R. et al. (1990; SerologicalMethods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D.E. et al. (1983; J. Exp. Med. 158:1211–1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labelled hybridisation or PCR probesfor detecting sequences related to polynucleotides encoding DevG20,DevG4, or DevG22 include oligo-labelling, nick translation,end-labelling or PCR amplification using a labelled nucleotide.

Alternatively, the sequences encoding DevG20, DevG4, or DevG22, or anyportions thereof may be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesise RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labellednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio).

Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, co-factors, inhibitors, magneticparticles, and the like.

Host cells transformed with nucleotide sequences encoding DevG20, DevG4,or DevG22 may be cultured under conditions suitable for the expressionand recovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors may be designed to containsignal sequences, which direct secretion of proteins through aprokaryotic or eukaryotic cell membrane. Other recombinant constructionsmay be used to join sequences encoding DevG20, DevG4, or DevG22 tonucleotide sequences encoding a polypeptide domain, which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilised metals, protein A domains that allow purification onimmobilised immunoglobulin, and the domain utilised in the FLAGextension/affinity purification system (Immunex Corp., Seattle, Wash.)The inclusion of cleavable linker sequences such as those specific forFactor XA or Enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and the desired protein may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing DevG20, DevG4, or DevG22 and a nucleic acidencoding 6 histidine residues preceding a Thioredoxine or anEnterokinase cleavage site. The histidine residues facilitatepurification on IMIAC (immobilised metal ion affinity chromatography asdescribed in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263–281))while the Enterokinase cleavage site provides a means for purifyingDevG20, DevG4, or DevG22 from the fusion protein. A discussion ofvectors which contain fusion proteins is provided in Kroll, D. J. et al.(1993; DNA Cell Biol. 12:441–453). In addition to recombinantproduction, fragments of DevG20, DevG4, or DevG22 may be produced bydirect peptide synthesis using solid-phase techniques (Merrifield J.(1963) J. Am. Chem. Soc. 85:2149–2154). Protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431A peptidesynthesiser (Perkin Elmer). Various fragments of DevG20, DevG4, orDevG22 may be chemically synthesised separately and combined usingchemical methods to produce the full length molecule.

Diagnostics and Therapeutics

The data disclosed in this invention show that the nucleic acids andproteins of the invention are useful in diagnostic and therapeuticapplications implicated, for example but not limited to, in metabolicdisorders like obesity, as well as related disorders such as adipositas,eating disorders, wasting syndromes (cachexia), pancreatic dysfunctions(such as diabetes mellitus), hypertension, pancreatic dysfunctions,arteriosclerosis, coronary artery disease (CAD), coronary heart disease,hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, cancer,e.g. cancers of the reproductive organs, sleep apnea, and other diseasesand disorders. Hence, diagnostic and therapeutic uses for the DevG20,DevG4, or DevG22 proteins of the invention are, for example but notlimited to, the following: (i) protein therapeutic, (ii) small moleculedrug target, (iii) antibody target (therapeutic, diagnostic, drugtargeting/cytotoxic antibody), (iv) diagnostic and/or prognostic marker,(v) gene therapy (gene delivery/gene ablation), (vi) research tools, and(vii) tissue regeneration in vitro and in vivo (regeneration for allthese tissues and cell types composing these tissues and cell typesderived from these tissues).

The nucleic acids and proteins of the invention are useful indiagnositic and therapeutic applications implicated in various diseasesand disorders described below and/or other pathologies and disorders.For example, but not limited to, cDNAs encoding the proteins of theinvention and particularly their human homologues may be useful in genetherapy, and the DevG20, DevG4, or DevG22 proteins of the invention andparticularly their human homologues may be useful when administered to asubject in need thereof. By way of non-limiting example, thecompositions of the present invention will have efficacy for treatmentof patients suffering from, for example, but not limited to, inmetabolic disorders like obesity, as well as related disorders such asadipositas, eating disorders, wasting syndromes (cachexia), pancreaticdysfunctions (such as diabetes mellitus), hypertension,arteriosclerosis, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancer, e.g. cancers of thereproductive organs, sleep apnea, and other diseases and disorders.

The nucleic acid encoding the DevG20, DevG4, or DevG22 protein of theinvention, or fragments thereof, may further be useful in diagnosticapplications, wherein the presence or amount of the nucleic acids or theproteins are to be assessed. These materials are further useful in thegeneration of antibodies that bind immunospecifically to the novelsubstances of the invention for use in therapeutic or diagnosticmethods.

For example, in one aspect, antibodies which are specific for DevG20,DevG4, or DevG22 may be used directly as an antagonist, or indirectly asa targeting or delivery mechanism for bringing a pharmaceutical agent tocells or tissue which express DevG20, DevG4, or DevG22. The antibodiesmay be generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimerical, single chain, Fab fragments, and fragments produced by a Fabexpression library. Neutralising antibodies, (i.e., those which inhibitdimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunised by injectionwith DevG20, DevG4, or DevG22 or any fragment or oligopeptide thereofwhich has immunogenic properties. Depending on the host species, variousadjuvants may be used to increase immunological response. Such adjuvantsinclude, but are not limited to, Freund's, mineral gels such asaluminium hydroxide, and surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, and dinitrophenol. Among adjuvants used in human, BCG(Bacille Calmette-Guerin) and Corynebacterium parvum are especiallypreferable. It is preferred that the peptides, fragments, oroligopeptides used to induce antibodies have an amino acid sequenceconsisting of at least five amino acids, and more preferably at least 10amino acids. It is preferable that they are identical to a portion ofthe amino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin in order to enhance theimmunogenicity.

Monoclonal antibodies to DevG20, DevG4, or DevG22 may be prepared usingany technique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Köhler, G. et al. (1975) Nature 256:495–497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31–42; Cote, R. J. etal. Proc. Natl. Acad. Sci. 80:2026–2030; Cole, S. P. et al. (1984) Mol.Cell Biol. 62:109–120).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851–6855; Neuberger, M. S. et al (1984) Nature312:604–608; Takeda, S. et al. (1985) Nature 314:452–454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to producesingle chain antibodies. Antibodies with related specificity, but ofdistinct idiotypic composition, may be generated by chain shuffling fromrandom combinatorial immunoglobulin libraries (Burton, D. R. (1991)Proc. Natl. Acad. Sci. 88:11120–3). Antibodies may also be produced byinducing in vivo production in the lymphocyte population or by screeningrecombinant immunoglobulin libraries or panels of highly specificbinding reagents as disclosed in the literature (Orlandi, R. et al.(1989) Proc. Natl. Acad. Sci. 86:3833–3837; Winter, G. et al. (1991)Nature 349:293–299).

Antibody fragments, which contain specific binding sites for DevG20,DevG4, or DevG22, may also be generated. For example, such fragmentsinclude, but are not limited to, the F(ab′)₂ fragments which can beproduced by Pepsin digestion of the antibody molecule and the Fabfragments which can be generated by reducing the disulfide bridges ofF(ab′)₂ fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse, W. D. et al. (1989)Science 254:1275–1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding and immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between the protein and its specific antibody. A two-site,monoclonal-based immunoassay utilising monoclonal antibodies reactive totwo non-interfering epitopes is preferred, but a competitive bindingassay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingDevG20, DevG4, or DevG22, or any fragment thereof, or antisensemolecules, may be used for therapeutic purposes. In one aspect,antisense molecules may be used in situations in which it would bedesirable to block the transcription of the mRNA. In particular, cellsmay be transformed with sequences complementary to polynucleotidesencoding DevG20, DevG4, or DevG22. Thus, antisense molecules may be usedto modulate protein activity, or to achieve regulation of gene function.Such technology is now well know in the art, and sense or antisenseoligomers or larger fragments, can be designed from various locationsalong the coding or control regions of sequences encoding DevG20, DevG4,or DevG22. Expression vectors derived from retroviruses, adenovirus,herpes or vaccinia viruses, or from various bacterial plasmids may beused for delivery of nucleotide sequences to the targeted organ, tissueor cell population. Methods, which are well known to those skilled inthe art, can be used to construct recombinant vectors, which willexpress antisense molecules complementary to the polynucleotides of thegene encoding DevG20, DevG4, or DevG22. These techniques are describedboth in Sambrook et al. (supra) and in Ausubel et al. (supra). Genesencoding DevG20, DevG4, or DevG22 can be turned off by transforming acell or tissue with expression vectors which express high levels ofpolynucleotide or fragment thereof which encodes DevG20, DevG4, orDevG22. Such constructs may be used to introduce untranslatable sense orantisense sequences into a cell. Even in the absence of integration intothe DNA, such vectors may continue to transcribe RNA molecules untilthey are disabled by endogenous nucleases. Transient expression may lastfor a month or more with a non-replicating vector and even longer ifappropriate replication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, e.g. DNA, RNA, or PNA molecules, to thecontrol regions of the genes encoding DevG20, DevG4, or DevG22, i.e.,the promoters, enhancers, and introns. Oligonucleotides derived from thetranscription initiation site, e.g., between positions −10 and +10 fromthe start site, are preferred. Similarly, inhibition can be achievedusing “triple helix” base-pairing methodology. Triple helix pairing isuseful because it cause inhibition of the ability of the double helix toopen sufficiently for the binding of polymerases, transcription factors,or regulatory molecules. Recent therapeutic advances using triplex DNAhave been described in the literature (Gee, J. E. et al. (1994) In;Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y.). The antisense molecules mayalso be designed to block translation of mRNA by preventing thetranscript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyse thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridisation of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage.Examples, which may be used, include engineered hammerhead motifribozyme molecules that can be specifically and efficiently catalyseendonucleolytic cleavage of sequences encoding DevG20, DevG4, or DevG22.Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridisation with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of nucleic acid molecules.These include techniques for chemically synthesising oligonucleotidessuch as solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding DevG20, DevG4, or DevG22. Such DNA sequences maybe incorporated into a variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesise antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues. RNA molecules may be modified toincrease intracellular stability and half-life. Possible modificationsinclude, but are not limited to, the addition of flanking sequences atthe 5′ and/or 3′ ends of the molecule or the use of phosphorothioate or2′ O-methyl rather than phosphodiesterase linkages within the backboneof the molecule. This concept is inherent in the production of PNAs andcan be extended in all of these molecules by the inclusion ofnon-traditional bases such as inosine, queosine, and wybutosine, as wellas acetyl-, methyl-, thio-, and similarly modified forms of adenine,cytidine, guanine, thymine, and uridine which are not as easilyrecognised by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods, which are well known in the art. Any of thetherapeutic methods described above may be applied to any suitablesubject including, for example, mammals such as dogs, cats, cows,horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of DevG20, DevG4, orDevG22, antibodies to DevG20, DevG4, or DevG22, mimetics, agonists,antagonists, or inhibitors of DevG20, DevG4, or DevG22. The compositionsmay be administered alone or in combination with at least one otheragent, such as stabilising compound, which may be administered in anysterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones. The pharmaceutical compositionsutilised in this invention may be administered by any number of routesincluding, but not limited to, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, or rectal means.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries, which facilitate processing of the activecompounds into preparations which, can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.). Pharmaceutical compositions for oraladministration can be formulated using pharmaceutically acceptablecarriers well known in the art in dosages suitable for oraladministration. Such carriers enable the pharmaceutical compositions tobe formulated as tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, suspensions, and the like, for ingestion by thepatient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including Arabic and tragacanth; andproteins such as gelatine and collagen. If desired, disintegrating orsolubilising agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate. Dragee cores may be used in conjunction with suitablecoatings, such as concentrated sugar solutions, which may also containgum Arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coating for product identification or to characterisethe quantity of active compound, i.e., dosage. Pharmaceuticalpreparations, which can be used orally, include push-fit capsules madeof gelatine, as well as soft, sealed capsules made of gelatine and acoating, such as glycerol or sorbitol. Push-fit capsules can containactive ingredients mixed with a filler or binders, such as lactose orstarches, lubricants, such as talc or magnesium stearate, and,optionally, stabilisers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquid,or liquid polyethylene glycol with or without stabilisers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances, which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilisers or agents who increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilising processes. Thepharmaceutical composition may be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulphuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. In other cases, the preferred preparation may be alyophilised powder which may contain any or all of the following: 1–50mM histidine, 0.1%–2% sucrose, and 2–7% mannitol, at a pH range of 4.5to 5.5, that is combined with buffer prior to use. After pharmaceuticalcompositions have been prepared, they can be placed in an appropriatecontainer and labelled for treatment of an indicated condition. Foradministration of DevG20, DevG4, or DevG22, such labelling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart. For any compounds, the therapeutically effective does can beestimated initially either in cell culture assays, e.g., of preadipocytecell lines, or in animal models, usually mice, rabbits, dogs, or pigs.The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans. A therapeutically effective dose refers to that amount of activeingredient, for example DevG20, DevG4, or DevG22 or fragments thereof,antibodies of DevG20, DevG4, or DevG22, to treat a specific condition.Therapeutic efficacy and toxicity may be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose therapeutically effective in 50% of the population)and LD50 (the dose lethal to 50% of the population). The dose ratiobetween therapeutic and toxic effects is the therapeutic index, and itcan be expressed as the ratio, LD50/ED50. Pharmaceutical compositions,which exhibit large therapeutic indices, are preferred. The dataobtained from cell culture assays and animal studies is used informulating a range of dosage for human use. The dosage contained insuch compositions is preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage from employed,sensitivity of the patient, and the route of administration. The exactdosage will be determined by the practitioner, in light of factorsrelated to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors, which may be takeninto account, include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy.

Long-acting pharmaceutical compositions may be administered every 3 to 4days, every week, or once every two weeks depending on half-life andclearance rate of the particular formulation. Normal dosage amounts mayvary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g,depending upon the route of administration. Guidance as to particulardosages and methods of delivery is provided in the literature andgenerally available to practitioners in the art. Those skilled in theart employ different formulations for nucleotides than for proteins ortheir inhibitors. Similarly, delivery of polynucleotides or polypeptideswill be specific to particular cells, conditions, locations, etc.

In another embodiment, antibodies which specifically bind DevG20, DevG4,or DevG22 may be used for the diagnosis of conditions or diseasescharacterised by or associated with over- or underexpression of DevG20,DevG4, or DevG22, or in assays to monitor patients being treated withDevG20, DevG4, or DevG22, agonists, antagonists or inhibitors. Theantibodies useful for diagnostic purposes may be prepared in the samemanner as those described above for therapeutics. Diagnostic assaysinclude methods, which utilise the antibody and a label to detectDevG20, DevG4, or DevG22 in human body fluids or extracts of cells ortissues. The antibodies may be used with or without modification, andmay be labelled by joining them, either covalently or non-covalently,with a reporter molecule. A wide variety of reporter molecules which areknown in the art may be used several of which are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringDevG20, DevG4, or DevG22 are known in the art and provide a basis fordiagnosing altered or abnormal levels of gene expression. Normal orstandard values for expression are established by combining body fluidsor cell extracts taken from normal mammalian subjects, preferably human,with antibodies to DevG20, DevG4, or DevG22 under conditions suitablefor complex formation. The amount of standard complex formation may bequantified by various methods, but preferably by photometry, means.Quantities of DevG20, DevG4, or DevG22 expressed in control and disease,samples from biopsied tissues are compared with the standard values.Deviation between standard and subject values establishes the parametersfor diagnosing disease.

In another embodiment of the invention, the polynucleotides specific forDevG20, DevG4, or DevG22 may be used for diagnostic purposes. Thepolynucleotides, which may be used, include oligonucleotide sequences,antisense RNA and DNA molecules, and PNAs. The polynucleotides may beused to detect and quantitate gene expression in biopsied tissues inwhich expression of DevG20, DevG4, or DevG22 may be correlated withdisease. The diagnostic assay may be used to distinguish betweenabsence, presence, and excess expression, and to monitor regulation ofgene expression levels during therapeutic intervention.

In one aspect, hybridisation with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding DevG20, DevG4, or DevG22 or closely related molecules, may beused to identify nucleic acid sequences which encode DevG20, DevG4, orDevG22. The specificity of the probe, whether it is made from a highlyspecific region, e.g., unique nucleotides in the 5′ regulatory region,or a less specific region, e.g., especially in the 3′ coding region, andthe stringency of the hybridisation or amplification (maximal, high,intermediate, or low) will determine whether the probe identifies onlynaturally occurring sequences encoding DevG20, DevG4, or DevG22,alleles, or related sequences. Probes may also be used for the detectionof related sequences, and should preferably contain at least 50% of thenucleotides from any of the DevG20, DevG4, or DevG22 encoding sequences.The hybridisation probes of the subject invention may be DNA or RNA andderived from the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQID NO:5 or from a genomic sequence including promoter, enhancerelements, and introns of the naturally occurring DevG20, DevG4, orDevG22 gene. Means for producing specific hybridisation probes for DNAsencoding DevG20, DevG4, or DevG22 include the cloning of nucleic acidsequences encoding DevG20, DevG4, or DevG22 or derivatives thereof intovectors for the production of mRNA probes. Such vectors are known in theart, commercially available, and may be used to synthesise RNA probes invitro by means of the addition of the appropriate RNA polymerases andthe appropriate labelled nucleotides. Hybridisation probes may belabelled by a variety of reporter groups, for example, radionuclidessuch as ³²P or ³⁵S, or enzymatic labels, such as alkaline phosphatasecoupled to the probe via avidin/biotin coupling systems, and the like.

Polynucleotide sequences specific for DevG20, DevG4, or DevG22 may beused for the diagnosis of conditions or diseases, which are associatedwith expression of DevG20, DevG4, or DevG22. Examples of such conditionsor diseases include, but are not limited to, pancreatic diseases anddisorders, including diabetes. Polynucleotide sequences specific forDevG20, DevG4, or DevG22 may also be used to monitor the progress ofpatients receiving treatment for pancreatic diseases and disorders,including diabetes. The polynucleotide sequences may be used in Southernor Northern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dip stick, pin, ELISA or chip assays utilisingfluids or tissues from patient biopsies to detect altered geneexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding DevG20, DevG4,or DevG22 may be useful in assays that detect activation or induction ofvarious metabolic diseases and disorders, including obesity, as well asrelated disorders such as described above. The nucleotide sequences maybe labelled by standard methods, and added to a fluid or tissue samplefrom a patient under conditions suitable for the formation ofhybridisation complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable have hybridised withnucleotide sequences in the sample, and the presence of altered levelsof nucleotide sequences in the sample indicates the presence of theassociated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of DevG20, DevG4, or DevG22, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, which is specific for DevG20, DevG4, or DevG22,under conditions suitable for hybridisation or amplification. Standardhybridisation may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease. Once disease is established and a treatment protocol isinitiated, hybridisation assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that, which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to metabolic diseases and disorders, including obesity, aswell as related disorders such as adipositas, eating disorders, wastingsyndromes (cachexia), pancreatic dysfunctions (such as diabetesmellitus), hypertension, arteriosclerosis, coronary heart disease,hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, cancer,e.g. cancers of the reproductive organs, sleep apnea, the presence of arelatively high amount of transcript in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the pancreatic diseases and disorders. Additionaldiagnostic uses for oligonucleotides designed from the sequencesencoding DevG20, DevG4, or DevG22 may involve the use of PCR. Sucholigomers may be chemically synthesised, generated enzymatically, orproduced from a recombinant source. Oligomers will preferably consist oftwo nucleotide sequences, one with sense orientation (5′.fwdarw.3′) andanother with antisense (3′.rarw.5′), employed under optimised conditionsfor identification of a specific gene or condition. The same twooligomers, nested sets of oligomers, or even a degenerate pool ofoligomers may be employed under less stringent conditions for detectionand/or quantification of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of DevG20,DevG4, or DevG22 include radiolabelling or biotinylating nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated (Melby, P. C. et al.(1993) J. Immunol. Methods, 159:235–244; Duplaa, C. et al. (1993) Anal.Biochem. 212:229–236). The speed of quantification of multiple samplesmay be accelerated by running the assay in an ELISA format where theoligomer of interest is presented in various dilutions and aspectrophotometric or colorimetric response gives rapid quantification.

In another embodiment of the invention, the nucleic acid sequences,which are specific for DevG20, DevG4, or DevG22, may also be used togenerate hybridisation probes, which are useful for mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome or to a specific region of the chromosome usingwell known techniques. Such techniques include FISH, FACS, or artificialchromosome constructions, such as yeast artificial chromosomes,bacterial artificial chromosomes, bacterial P1 constructions or singlechromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood Rev.7:127–134, and Trask, B. J. (1991) Trends Genet. 7:149–154. FISH (asdescribed in Verma et al. (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York, N.Y.) may be correlated with otherphysical chromosome mapping techniques and genetic map data. Examples ofgenetic map data can be found in the 1994 Genome Issue of Science(265:1981f). Correlation between the location of the gene encodingDevG20, DevG4, or DevG22 on a physical chromosomal map and a specificdisease, or predisposition to a specific disease, may help to delimitthe region of DNA associated with that genetic disease.

The nucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier, or affectedindividuals. In situ hybridisation of chromosomal preparations andphysical mapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localised by genetic linkage toa particular genomic region, for example, AT to 11 q22–23 (Gatti, R. A.et al. (1988) Nature 336:577–580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequences of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, DevG20, DevG4, or DevG22, theircatalytic or immunogenic fragments or oligopeptides thereof, can be usedfor screening libraries of compounds, e.g. peptides or low-molecularweight organic molecules, in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between theprotein and the agent tested, may be measured.

Another technique for drug screening, which may be used, provides forhigh throughput screening of compounds having suitable binding affinityto the protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to DevG20, DevG4, or DevG22 largenumbers of different test compounds, e.g. small molecules, aresynthesised on a solid substrate, such as plastic pins or some othersurface. The test compounds are reacted with the proteins, or fragmentsthereof, and washed. Bound proteins are then detected by methods wellknown in the art. Purified proteins can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralising antibodies can be used to capture thepeptide and immobilise it on a solid support. In another embodiment, onemay use competitive drug screening assays in which neutralisingantibodies capable of binding DevG20, DevG4, or DevG22 specificallycompete with a test compound for binding DevG20, DevG4, or DevG22. Inthis manner, the antibodies can be used to detect the presence of anypeptide, which shares one or more antigenic determinants with DevG20,DevG4, or DevG22. In additional embodiments, the nucleotide sequenceswhich encode DevG20, DevG4, or DevG22 may be used in any molecularbiology techniques that have yet to be developed, provided the newtechniques rely on properties of nucleotide that are currently known,including, but not limited to, such properties as the triplet geneticcode and specific base pair interactions.

THE FIGURES SHOW

FIG. 1 shows the average increase of starvation resistance of HD-EP(X)10478 and HD-EP(X)31424 flies (Drosophila melanogaster; in the Figure,referred to as 10478 and 31424) by ecotopic expression using ‘FB- orelav-Gal4 driver’ (in comparison to wildtype flies (Oregon R); FB standsfor fat body; elav-Gal4 stands for elevated Gal4). The average valuesfor surviving flies (,average survivors) are given in % per time point(shown on the horizontal line as time of starvation; 8 hours (8 h) to 72hours (72 h)) are shown. See Examples for a more detailed description.

FIG. 2 shows the increase of triglyceride content of HD-EP(X)10478 andHD-EP(X)31424 flies by ectopic expression using “FB- or elav-Gal4driver” (in comparison to wildtype flies (Oregon R)). Standard deviationof the measurements is shown as thin bars. Triglyceride content of thefly populations is shown in ug/mg wet weight (wt) of a fly (vertical).

FIG. 3 shows the molecular organisation of the DevG20 locus.

FIG. 4A shows the nucleic acid sequence (SEQ ID NO:7) encoding theDrosophila DevG20 protein.

FIG. 4B shows the protein sequence (SEQ ID NO:8) of the DrosophilaDevG20 encoded by the nucleic acid sequence shown in FIG. 4A.

FIG. 4C shows the nucleic acid sequence (SEQ ID NO:1) of the humanDevG20 homolog protein encoding the Homo sapiens hypothetical proteinwith Genbank Accession Number NM_(—)030810.1 (MGC3178).

FIG. 4D shows the human DevG20 protein sequence (SEQ ID NO:2) (GenBankAccession Number NP_(—)110437.1) encoded by the nucleic acid sequenceshown in FIG. 4C.

FIG. 5 shows the BLASTP (versus the non-redundant composite database)identity search result for Drosophila DevG20 protein (SEQ ID NO:8) andthe human DevG20 protein (SEQ ID NO:2; GenBank Accession NumberNP_(—)110437.1), referred to as hG20 in the Figure. The middle sequenceof the alignment shows identical amino acids in the one-letter code andconserved as +. Gaps in the alignment are represented as −.

FIG. 6 shows the expression of DevG20 in mammalian tissues.

FIG. 6A shows the real-time PCR analysis of DevG20-like expression indifferent wildtype mouse tissues. The relative RNA-expression is shownon the left hand side, the tissues tested are given on the horizontalline (for example, pancreas (‘pancre’), white adipose tissue (‘WA’),brown adipose tissue (‘BA’), muscle (‘muscl’), liver (‘liv’),hypothalamus (‘hypothalam’), cerebellum (‘cerebell’), cortex (‘corte’),midbrain (‘midbra’); small intestine (‘s. intestine’), heart (‘hear’),lung (‘lun’), spleen (‘splee’), kidney (‘kidne’), and bone marrow (‘b.marrow’).

FIG. 6B shows the real-time PCR analysis of DevG20-like expression indifferent mouse models (wildtype mice (‘wt’)— bars with light greyshading; fasted mice—bars with dark grey shading, obese mice (‘ob/ob’),white bar) in different tissues (white adipose tissue (‘WA’), brownadipose tissue (‘BA’), muscle (‘musc’), liver (‘liv’), pancreas(‘pancre’), hypothalamus (‘hypothalam’), cerebellum (‘cerebell’), cortex(‘corte’), midbrain (‘midbra’); small intestine (‘s. intestine’), heart(‘hear’), lung (‘lun’), spleen (‘splee’), kidney (‘kidne’), and bonemarrow (‘b. marrow’).

FIG. 6C shows the real-time PCR analysis of DevG20-like expressionduring the differentiation of 3T3-L1 cells from pre-adipocytes to matureadipocytes. The relative RNA-expression is shown on the left hand side,the days of differention are shown on the horizontal line (d0=day 0,start of the experiment, until d10=day 10).

FIG. 7 shows the relative increase of triglyceride content of EP(2)0646,EP(2)2188, and EP(2)2517 flies caused by homozygous viable integrationof the P-vector (in comparison to wildtype flies (EP-control)). Standarddeviation of the measurements is shown as thin bars. Triglyceridecontent of the fly populations is shown as ration TG/Protein content.

FIG. 8 shows the molecular organisation of the DevG4 gene locus.

FIG. 9A shows the nucleic acid sequence (SEQ ID NO:9) encoding theDrosophila DevG4 protein.

FIG. 9B shows the Drosophila DevG4 protein sequence (SEQ ID NO:10)encoded by the mRNA shown in FIG. 9A.

FIG. 9C shows the nucleic acid sequence (SEQ ID NO:3) encoding the humanDevG4 homolog (Homo sapiens ATP-binding cassette, sub-family C(CFTR/MRP), member 4, also referred to as ABCC4 and MPR4; GenBankAccession Number NM_(—)005845).

FIG. 9D shows the protein sequence (SEQ ID NO:4; GenBank AccessionNumber NP_(—)005836.1) of the human DevG4 homolog.

FIG. 10 shows protein domains (black boxes) of the human DevG4 protein.

FIG. 11 shows the comparison of DevG4 protein domains of differentspecies (human, hMRP4′, mouse (only shown in FIG. 11D, mMRP4), andDrosophila (DevG4)). Gaps in the alignment are represented as −. Thealignment was produced using the multisequence alignment program ofClustal V software (Higgins, D. G. and Sharp, P. M. (1989). CABIOS, vol.5, no. 2, 151–153.).

(A) Alignment of the ABC-membrane I domains. The identity of amino acidsof Drosophila DevG4 and human DevG4 (hMRP4) is 41% and the similarity63%.

(B) Alignment of the ABC-tran I domains. The identity of amino acids ofDrosophila DevG4 and human DevG4 (hMRP4) is 56% and the similarity 75%.

(C) Alignment of the ABC-membrane II domains. The identity of aminoacids of Drosophila DevG4 and human DevG4 (hMRP4) is 42% and thesimilarity 60%.

(D) Alignment of the ABC-tran II domains. The identity of amino acids ofDrosophila DevG4 and human DevG4 (hMRP4) is 69% and the similarity 86%.Human and mouse ABC-tran II are almost identical.

FIG. 12 shows the expression of DevG4 in mammalian tissues.

FIG. 12A shows the real-time PCR analysis of DevG4 (MRP4) expression indifferent wildtype mouse tissues (pancreas (‘pancre’), white adiposetissue (‘WA’), brown adipose tissue (‘BA’), muscle (‘muscl’), liver(‘liv’), hypothalamus (‘hypothalam’), cerebellum (‘cerebell’), cortex(‘corte’), midbrain (‘midbra’); small intestine (‘s. intestine’), heart(‘hear’), lung (‘lun’), spleen (‘splee’), kidney (‘kidne’), and bonemarrow (‘b. marrow’). The relative RNA-expression is shown on the lefthand side, the tissues tested are given on the horizontal line.

FIG. 12B shows the real-time PCR analysis of DevG4 (MRP4) expression indifferent mouse models (wildtype mice (‘wt’), fasted mice, obese mice(‘ob/ob’)) in different tissues (white adipose tissue (‘WA’), brownadipose tissue (‘BA’), muscle (‘muscl’), liver (‘liv’), pancreas(‘pancre’), hypothalamus (‘hypothalam’), cerebellum (‘cerebell’), cortex(‘corte’), midbrain (‘midbra’); small intestine (‘s. intestine’), heart(‘hear’), lung (‘lun’), spleen (‘splee’), kidney (‘kidne’), and bonemarrow (‘b. marrow’).

FIG. 12C shows the real-time PCR analysis of DevG4 (MRP4) expressionduring the differentiation of 3T3-L1 cells from pre-adipocytes to matureadipocytes. The relative RNA-expression is shown on the left hand side,the days of differention are shown on the horizontal line (d0=day 0,start of the experiment, until d10=day 10).

FIG. 13 shows the relative increase of triglyceride content ofHD-EP(2)20388 and EP(2)2482 flies caused by homozygous viableintegration of the P-vector (in comparison to wildtype flies(EP-control)). Standard deviation of the measurements is shown as thinbars. Triglyceride content of the fly populations is shown as ratioTG/Protein content in percent (%).

FIG. 14 shows the molecular organisation of the DevG22 gene locus.

FIG. 15A shows the nucleic acid sequence (SEQ ID NO:11) encoding theDrosophila DevG22 protein.

FIG. 15B shows the protein sequence (SEQ ID NO:12) of the DrosophilaDevG22 protein.

FIG. 15C shows the nucleic acid sequence (SEQ ID NO:5) encoding thehuman DevG22 homolog (Homo sapiens ATP-binding cassette, sub-family G(WHITE), member 1 protein; GenBank Accession Number XM_(—)009777).

FIG. 15D shows the protein sequence (SEQ ID NO:6) of the human DevG22homolog (Homo sapiens ATP-binding cassette, sub-family G (WHITE), member1 protein; GenBank Accession Number XP_(—)009777.3).

FIG. 16 shows protein domain (black box) of the DevG22 protein.

FIG. 17 shows the alignment of human, mouse and fly DevG22 proteins(White-like ABC transporters). White-like ABC transporters only have asingle ABC-tran protein domain. Drosophila DevG22 is 36% identical and52% similar to human DevG22 (hWhite; ABC8, ABCG1, GenBank AccessionNumber XM_(—)009777). Drosophila DevG22 is 36% identical and 51% similarto mouse DevG22 (mWhite; GenBank Accession Number NP_(—)033723). Humanand mouse DevG22 proteins show 95% identity and 96% similarity.

FIG. 18 shows the expression of DevG22 in mammalian tissues.

FIG. 18A shows the real-time PCR analysis of DevG22 expression indifferent wildtype mouse tissues (pancreas (‘pancre’), white adiposetissue (‘WA’), brown adipose tissue (‘BA’), muscle (‘muscl’), liver(‘liv’), hypothalamus (‘hypothalam’), cerebellum (‘cerebell’), cortex(‘corte’), midbrain (‘midbra’); small intestine (‘sm. testine’), heart(‘hear’), lung (‘lun’), spleen (‘splee’), kidney (‘kidne’), and bonemarrow (‘b. marrow’).

FIG. 18B shows the real-time PCR analysis of DevG22 expression indifferent mouse models (wildtype mice (‘wt’), fasted mice, obese mice(‘ob/ob’)) in different tissues (white adipose tissue (‘WA’), brownadipose tissue (‘BA’), muscle (‘muscl’), liver (‘liv’), pancreas(‘pancre’), hypothalamus (‘hypothalam’), cerebellum (‘cerebell’), cortex(‘corte’), midbrain (‘midbra’); small intestine (‘s. intestine’), heart(‘hear’), lung (‘lun’), spleen (‘splee’), kidney (‘kidne’), and bonemarrow (‘b. marrow’).

FIG. 18C shows the real-time PCR analysis of DevG22 expression duringthe differentiation of 3T3-L1 cells from pre-adipocytes to matureadipocytes.

EXAMPLES

A better understanding of the present invention and of its manyadvantages will be evident from the following examples, only given byway of illustration.

Example 1 Isolation of EP-Lines That Have a Novel Function in EnergyHomeostasis Using a Functional Genetic Screen

In order to isolate genes with a function in energy homeostasis severalthousand EP-lines were crossed against two “Gal4-driver” lines thatdirect expression of Gal4 in a tissue specific manner. Two different“driver”-lines were used in the screen: (i) expressing Gal4 mainly inthe fatbody (FB), (ii) expressing Gal4 in neurons (elav) (FIG. 1). Aftercrossing the “driver”-line to the EP-line, an endogenous gene may beactivated in fatbody or neurons respectively. For selection of relevantgenes affecting energy homeostasis, the offspring of that cross wasexposed to starvation conditions after six days of feeding. Wildtypeflies show a constant starvation resistance. EP-lines with significantlychanged starvation resistance were selected as positive candidates.

Example 2 HD-EP(X)10478 and HD-EP(X)31424 Flies Show SignificantStarvation Resistance When Driven in the Fatbody or Neurons

Ectopic expression of the EP-lines HD-EP(X)10478 and HD-EP(X)31424, bothhomozygous viable integrations in the chromosomal region 10D4–10D6,under the control of the “FB-driver” and “elav-driver” caused asignificant starvation resistance in comparison to wildtype flies(Oregon R, see FIG. 1). Hundred flies offfspring of a cross or line wereanalysed under starvation conditions. Survivors per time point are shownin FIG. 1. After 24 hours of starvation HD-EP(X)10478 and 31424 flies incombination with both “drivers” show 80–100% more survivors than thewildtype Oregon R. After 48 hours of starvation, almost no wildtypeflies are still alive. In contrast, after 48 hours of starvation, about20% of the population of HD-EP(X)10478 and 31424 in combination withboth “drivers” are alive which is a significant increase. Few flies ofHD-EP(X) 10478 and 31424 in combination with both “drivers” stillsurvive after 72 hours of starvation where normally no wildtype fliesare alive. Therefore, ectopic expression via HD-EP(X)10478 andHD-EP(X)31424 in the fatbody and neurons of Drosophila melanogasterleads to significant starvation resistance.

Example 3 Triglyceride Content is Increased by Ectopic Expression viaHD-EP(X) 10478 and HD-EP(X)31424 in the Fatbody and Weaker in theNeurons

Starvation resistance can have its origin due to changes in energyhomeostasis, e.g., reduction of energy consumption and/or increase instorage of substances like triglycerides. Triglycerides are the mostefficient storage for energy in cells. Therefore the content oftriglycerides of a pool of flies with the same genotype was analysedusing an triglyceride assay.

For determination of triglyceride content of flies, several aliquots ofeach time ten females of HD-EP(X)10478 and HD-EP(X)31424,HD-EP(X)10478/FB-Gal4 and HD-EP(X)31424/FB-Gal4, HD-EP(X)10478/elav-Gal4and HD-EP(X)31424/elav-Gal4 and Oregon R were analysed. Flies wereincubated for 5 min at 90° C. in an aqueous buffer using a waterbath,followed by hot extraction. After another 5 min incuabation at 90° C.and mild centrifugation, the triglyceride content of the flies extractwas determined using Sigma Triglyceride (INT 336-10 or -20) assay bymeasuring changes in the optical density according to the manufacturer'sprotocol. As a reference fly mass was measured on a fine balance beforeextraction procedure.

The result of the triglyceride contents analysis is shown in FIG. 2. Theaverage increase of triglyceride content of HD-EP(X)10478 and 31424flies in combination with the “FB- and elav Gal4-driver” lines is shownin comparison to wildtype flies (Oregon R) and the HD-EP(X)10478 and31424 integrations alone. Standard deviations of the measurments areshown as thin bars. Triglyceride content of the different flypopulations is shown in μg/mg wet weight (wt.) of a fly. In each assayten females of the offspring of a cross or line were analysed in thetriglyceride assay after feeding that offspring for six days. The assaywas repeated several times. Wildtype flies show a constant triglyceridelevel of 30 to 45 μg/mg wet weight of a fly. HD-EP(X)10478 and 31424flies show a similar or slightly lower triglyceride content thanwildtype. In contrast, HD-EP(X)10478 and 31424 flies in combination withboth “drivers” show an average increase up to 1.8-fold of 55 to 72 μg/mgwet wt. in comparison to wildtype (Oregon R) flies and the HD-EP(X)10478and 31424 integration alone. Therefore, gain of a gene activity in thelocus 10D4-6 is responsible for changes in the metabolism of the energystorage triglycerides.

Ectopic expression of genomic Drosophila sequences using FB-Gal4 causedan average 1.8-fold increase of triglyceride content in comparison towildtype flies (Oregon R). HD-EP(X)10478 and HD-EP(X)31424 under thecontrol of the “elav-Gal4-driver” caused a weaker increase oftriglyceride content. Therefore ectopic expression via HD-EP(X)10478 andHD-EP(X)31424 in the fatbody of Drosophila melanogaster leads to asignificant increase of the energy storage triglyceride and thereforerepresents an obese fly model. The increase of triglyceride content bygain of a gene function suggests a gene activity in energy homeostasisin a dose dependent and tissue specific manner that controls the amountof energy stored as triglycerides.

Example 4 Measurement of Triglyceride Content of Homozygous Flies(EP(2)0646, EP(2)2188, EP(2)2517, HD-EP(2)20388, EP(2)2482)

Triglycerides are the most efficient storage for energy in cells. Inorder to isolate genes with a function in energy homeostasis, severalthousand EP-lines were tested for their triglyceride content after aprolonged feeding period. Lines with significantly changed triglyceridecontent were selected as positive candidates for further analysis. Inthis invention, the content of triglycerides of a pool of flies with thesame genotype after feeding for six days was analysed using atriglyceride assay. For determination of triglyceride content, severalaliquots of each time 10 males of the offspring of a cross or line wereanalysed after feeding the offspring for six days. Fly mass was measuredon a fine balance as a reference. Flies were extracted inmethanol/chloroform (1:1) and an aliquot of the extract was evaporatedunder vacuum. Lipids were emulsified in an aqueous buffer with help ofsonification. Triglyceride content was determined using Sigma INT 336-10or -20 assay by measuring changes in the optical density according tothe manufacturer's protocol.

Improving and simplifying the determination of triglyceride content offlies, In each assay ten males of the offspring of a cross or line wereanalysed in the triglyceride assay after feeding that offspring for sixdays; the assay was repeated several times. Flies were incubated for 5min at 90° C. in an aqueous buffer using a waterbath, followed by hotextraction. After another 5 min incubation at 90° C. and mildcentrifugation, the triglyceride content of the flies extract wasdetermined using Sigma Triglyceride (INT 336-10 or -20) assay bymeasuring changes in the optical density according to the manufacturer'sprotocol. As a reference protein content of the same extract wasmeasured using BIO-RAD DC Protein Assay according to the manufacturer'sprotocol.

Wildtype flies show constantly a triglyceride level of 11 to 23 μg/mgwet weight of a fly. EP(2)0646, EP(2)2188, EP(2)2517, and HD-EP(2)20388,and EP(2)2482 homozygous flies show constantly a higher triglyceridecontent than the wildtype (FIGS. 7 and 13). In contrast, EP(2)0646,EP(2)2188, EP(2)2517, HD-EP(2)20388, and EP(2)2482 flies in combinationwith both “drivers” show sometimes only a slightly increase (2.1- to2.3-fold of 49 to 53 μg/mg wet wt) in comparison to the wildtype (OregonR) (not shown). Therefore, the loss of gene activity in the loci, wherethe P-vector of EP(2)0646, EP(2)2188, EP(2)2517, HD-EP(2)20388, andEP(2)2482 flies is homozygous viably integrated, is responsible forchanges in the metabolism of the energy storage triglycerides, thereforerepresenting in both cases an obese fly model. The increase oftriglyceride content due to the loss of a gene function suggestspotential gene activities in energy homeostasis in a dose dependentmanner that controls the amount of energy stored as triglycerides.

Example 5 Identificiation of the Genes

DevG20 (PDI)

Nucleic acids encoding the DevG20 protein of the present invention wereidentified using plasmid-rescue technique. Genomic DNA sequences ofabout 1 kb were isolated that are localised directly 3′ to HD-EP(X)10478or HD-EP(X)31424 integrations. Using those isolated genomic sequencespublic databases like Berkeley Drosophila Genome Project (GadFly) werescreened thereby confirming the integration side of HD-EP(X)10478 andHD-EP(X)31424 and nearby localised endogenous genes (FIG. 3). FIG. 3shows the molecular organisation of the DevG20 locus. Genomic DNAsequence is represented by the assembly AE003487 as a black line thatincludes the integration sites of EP(X)1503, HD-EP(X)10478 andHD-EP(X)31424. Numbers represent the coordinates of AE003487 genomicDNA, the predicted genes and the EP-vector integration sites. Arrowsrepresent the direction of ectopic expression of endogenous genescontrolled by the Gal4 promoters in the EP-vectors. Predicted exons ofgenes CG2446 and CG 1837 are shown as grey bars. Using plasmid rescuemethod about 1 kb genomic DNA sequences that are directly localised 3′of the HD-EP(X)10478 and HD-EP(X)31424 integration sites were isolated.Using the 1 kb plasmid rescue DNA public DNA sequence databases werescreened thereby identifying the integration sites of HD-EP(X)10478 andHD-EP(X)31424.

HD-EP(X)10478 and HD-EP(X)31424 are integrated in the predicted geneCG2446 that is represented by the EST clots 241_(—)2–4 but their Gal4promoters direct ectopic expression of endogenous genes in the oppositedirection in respect to the direction of CG2446 expression. About 2 kb3′ of HD-EP(X)10478 and HD-EP(X)31424 integration sites the predictedgene CG1837 is localized that corresponds to est clot 3553_(—)14 andcould be expressed ectopically using “FB- and elav-Gal4-drivers”. Theectopic expression of CG1837 in the fatbody or weaker in neurons leadsto increase of triglyceride content in flies.

HD-EP(X)10478 is inserted into the first predicted exon of CG2446 thatcorresponds to the EST clot 241_(—)2–4 in antisense orientation. Gal4promoter region of HD-EP(X)10478 drives expression in the oppositedirection than CG2446 is expressed therefore could drive the ectopicexpression of another endogenous gene. HD-EP(X)31424 is inserted in thefirst predicted exon of CG2446 and its Gal4 promoter drives expressionin the opposite direction in comparison to CG2446 expression. Adifferent endogenous gene CG1837 corresponding to EST clot 3553_(—)14 islocalized 2180 base pairs 3′ in sense direction of both EP-integrations.CG1837 can be expressed ectopically via HD-EP(X)10478 and HD-EP(X)31424,leading to obesity.

DevG4 (MRP4)

Nucleic acids encoding the DevG4 protein of the present invention wereidentified using plasmid-rescue technique. Genomic DNA sequences ofabout 0.8 kb were isolated that are localised directly 3′ to theEP(2)0646, EP(2)2517 and EP(2)2188 integration. Using those isolatedgenomic sequences public databases like Berkeley Drosophila GenomeProject (GadFly) were screened thereby confirming the integration sideof 0646, EP(2)2517 and EP(2)2188 and nearby localised endogenous genes(FIG. 8). FIG. 8 shows the molecular organisation of the DevG4 locus. InFIG. 8, genomic DNA sequence is represented by the assembly as a dottedblack line (17.5 kb, starting at position 8256000 on chromosome 2L) thatincludes the integration sites of 0646, EP(2)2517 and EP(2)2188(arrows). Numbers represent the coordinates of the genomic DNA. Arrowsrepresent the direction of ectopic expression of endogenous genescontrolled by the Gal4 promoters in the EP-vectors. Transcribed DNAsequences (ESTs and clots) are shown as bars in the lower two lines.

Predicted exons of gene CG7627 (GadFly) are shown as green bars andintrons as grey bars.

0646, EP(2)2517 and EP(2)2188 are integrated directly 5′ of the EST Clot6022_(—)1 in antisense orientation. Clot 6022_(—)1 represents a cDNAclone meaning that is showing that the DNA sequence is expressed inDrosophila. Clot 6022_(—)1 sequence overlaps with the sequence of thepredicted gene CG7627 therefore Clot 6022_(—)1 includes the 5′ end ofDevG4 gene and EP(2)0646 and EP(2)2517 are homozygous viably integratedin the promoter of DevG4. Using the 0.8 kb plasmid rescue DNA, publicDNA sequence databases were screened thereby identifying the integrationsites of EP(2)0646 and EP(2)2517. It was found that EP(2)0646 andEP(2)2517 are integrated in the promoter of the gene with GadFlyAccession Number CG7627 that is also represented by the EST clot6022_(—)1. The Gal4 promoters of should direct ectopic expression ofendogenous genes in the opposite direction in respect to the directionof CG7627 expression. Therefore, expression of the CG7627 could beeffected by homozygous viable integration of EP(2)0646, EP(2)2517 andEP(2)2188 leading to increase of the energy storage triglycerides

DevG22

FIG. 14 shows genomic DNA sequence represented by the assembly as adotted black line (15 kb, starting at position 171400.5 on chromosome2L) that includes the integration site of EP(2)20388 (arrow). Numbersrepresent the coordinates of the genomic DNA. Arrows represent thedirection of ectopic expression of endogenous genes controlled by theGal4 promoters in the EP-vectors. Transcribed DNA sequences (ESTs andclots) are shown as green bars in another line. Predicted exons of genewith GadFly Accession Number CG17646 are shown as green bars and intronsas grey bars. It was found that DevG22 encodes for a novel gene that ispredicted by GadFly sequence analysis programs as CG17646. Using plasmidrescue method about 0.6 kb genomic DNA sequences that are directlylocalised 3′ of the EP(2)20388 integration site were isolated. Using the0.6 kb plasmid rescue DNA, public DNA sequence databases were screenedthereby identifying the integration site of EP(2)20388. EP(2)20388 isintegrated directly 5′ of the EST SD03967 in sense orientation. SD03967represents a cDNA clone meaning that its DNA sequence is expressed inDrosophila. SD03967 sequence overlaps with the 5′ sequence of thepredicted gene CG 17646 therefore SD03967 includes the 5′ and the 3′ endof DevG22 gene. The 3′ end of SD03967 does not overlap with CG17646sequence therefore the cDNA of DevG22 might be even longer than shown inFIG. 14. EP(2)20388 is integrated in the promoter of the gene CG17646that is also represented by EST SD03967; its Gal4 promoter should directectopic expression of CG 17646. Therefore, expression of the CG 17646could be effected by homozygous viable integration of EP(2)20388 leadingto increase of the energy storage triglycerides.

Example 6 Analysis of DevG20

DevG20 encodes for a novel gene that is predicted by GadFly sequenceanalysis programs and isolated EST clones. Neither phenotypic norfunctional data are available in the prior art for the novel geneCG1837, referred to as DevG20 in the present invention. The presentinvention is describing the nucleic acid sequence of DevG20, as shown inFIG. 4A, SEQ ID NO:1.

The present invention is describing a polypeptide comprising the aminoacid sequence of SEQ ID NO:2, as presented using the one-letter code inFIG. 4B. DevG20 is 416 amino acids in length. An open reading frame wasidentified by beginning with an ATP initiation codon at nucleotide 37and ending with a CAC stop codon at nucleotide 1284 (FIG. 4B).

The predicted amino acid sequence was searched in the publicly availableGenBank database. In search of sequence databases, it was found, forexample, that DevG20 has 60% homology with human hGRP58 protein, apotential 58 kDa glucose regulated protein of 324 amino acids (GenBankAccession Number NP_(—)110437.1; identical to former Accession NumbersAAH01199 and BC001199) (see FIGS. 4C and 4D; SEQ ID NO:1 and 2). Inparticular, Drosophila DevG20 and human hGRP58 protein share 60%homology (see FIG. 5), starting between amino acid 84 and 407 of DevG20(and amino acids 1 to 316 of hGRP58). hGRP58 protein is homologous to amouse protein encoded by the cDNA clone 601333564F1 NCI_CGAP_Mam6,identified using tblastp sequence comparison of a protein withtranslated mouse EST clones.

Using InterPro protein analysis tools, it was found, for example, thatthe DevG20 protein has at least three Thioredoxin protein motifs and anendoplasmic reticulum target sequence. These motifs and targetingsequencing are also found in glucose-regulated proteins and Proteindisulfide isomerases. Glucose regulated proteins and Protein disulfideisomerases are chaperones that are involved in many different processeslike lipoprotein assembly at the endoplasmic reticulum.

DevG20 encodes for a novel protein that is homologous to the family ofprotein disulfide isomerases or glucose regulated proteins. Based uponhomology, DevG20 protein of the invention and each homologous protein orpeptide may share at least some activity.

Example 7 Analysis of DevG4

As described above, DevG4 is encoded by GadFly Accession Number CG7627.The nucleic acid sequence of Drosophila DevG4, as shown in FIG. 9A, SEQID NO:9. The present invention is describing a polypeptide comprisingthe amino acid sequence of SEQ ID NO:10, as presented using theone-letter code in FIG. 9B. Drosophila DevG4 protein is 1355 amino acidsin length. An open reading frame was identified beginning with an ATPinitiation codon at nucleotide 158 and ending with a stop codon atnucleotide 4225. Drosophila DevG4 has additional 28 amino acids at theN-terminus without changing the frame in comparison to the predictedCG7627 protein after combining Clot 6022_(—)1 and CG7627 cDNA sequences.

The predicted amino acid sequence was searched in the publicly availableGenBank (NCBI) database. The search indicated, that Drosophila DevG4 hasabout 40% identity with human MRP4 (MOAT-B) protein, a ATP-bindingcassette (ABC) transporter protein of 1325 amino acids (AccessionNumber: NP_(—)005836; SEQ ID NO:10) (see FIG. 9C). In particular,Drosophila DevG4 and human homolog DevG4 (hMRP4) proteins share about80% homology (see FIG. 9D), starting between amino acid 8 and 1330 ofDevG4 (and amino acids 7 to 1277 of hMRP4).

Since the protein domains found in member of the ABC superfamily arehighly conserved, a comparison (Clustal X 1.8) between the four proteindomains of Drosophila DevG4 with human and mouse homolog proteins wasconducted (see FIG. 11). We found that human and mouse (sequence is onlypartially available) MRP4 as closest homologous proteins to theDrosophila DevG4 protein. Using InterPro protein analysis tools, it wasfound, that the DevG4 protein has at least 4 four protein motifs domains(FIG. 10). These motifs and targeting sequencing are found throughoutthe whole ABC transporter superfamliy. ABC transporters are membranespanning proteins that are involved in many different transportprocesses. FIG. 11A shows the alignment of the ABC-membrane I domains.The identity of amino acids of Drosophila DevG4 and human hMRP4 is 41%and the similarity of the sequence is 63%. FIG. 11B shows the alignmentof the ABC-tran I domains. The identity of amino acids of DrosophilaDevG4 and human hMRP4 is 56% and the similarity 75%. No mouse sequenceis available. FIG. 11C shows the alignment of the ABC-membrane IIdomains. The identity of amino acids of Drosophila DevG4 and human hMRP4is 42% and the similarity 60%. No mouse sequence is available. FIG. 11Dshows the alignment of the ABC-tran II domains. No mouse sequence isavailable. The identity of amino acids of Drosophila DevG4 and humanhMRP4 is 69% and the similarity 86%. Human and mouse ABC-tran II domainsare almost identical.

Based upon homology, Drosophila DevG4 protein and each homologousprotein or peptide may share at least some activity. The DevG4 proteinhas two characteristic ABC-membrane domains, a six transmembrane helicalregion (labeled ‘ABC_membrane’ in FIG. 10, ABC transporter transmembraneregion) which anchors the protein in cell membranes. In addition, DevG4has two ABC-transporter domains of several hundred amino acid residues(labeled ‘ABC-tran’ in FIG. 10, ABC transporter), including anATP-binding site. Proteins of the ABC family are membrane spanningproteins associated with a variety of distinct biological processes inboth prokaryotes and eukaryotes, for example in transport processes suchas active transport of small hydrophilic molecules across thecytoplasmic membrane. Furthermore, a single MMR-HSR1 domain (GTPase ofunknown function, light grey square box in FIG. 4A) was identified inDevG4. FIG. 10 shows the has a single characteristic ABC-transporterdomain (‘ABC trans’) of the DevG22 protein.

Example 8 Analyis of DevG22

As discussed above, Drosophila DevG22 protein is encoded GadFlyaccession number CG 17646. The present invention is describing thenucleic acid sequence of DevG22, as shown in FIG. 15A, SEQ ID NO:11. Thepresent invention is describing a polypeptide comprising the amino acidsequence of SEQ ID NO:12, as presented using the one-letter code in FIG.15B. Drosophila DevG22 protein is 627 amino acids in length. An openreading frame was identified beginning with an ATP initiation codon atnucleotide 576 and ending with a stop codon at nucleotide 2459.

The predicted amino acid sequence was searched in the publicly availableGenBank database. In search of sequence databases, it was found, forexample, that DevG22 has almost 40% identity with human White (ABC8,ABCG1) protein, a ATP-binding cassette (ABC) transporter protein of 674amino acids (GenBank Accession Number XP_(—)009777.3; see FIGS. 15C and15D; SEQ ID NO:5 and SEQ ID NO:6). In particular, Drosophila DevG22 andthe human homolog protein share about 70% homology (see FIG. 17),starting between amino acid 57 and 622 of Drosophila DevG22 (and aminoacids 27 to 507 of human DevG22-hWhite).

Using InterPro protein analysis tools, it was found that the DevG22protein has at least 1 one protein motif (FIG. 16). The White-likesubfamily of ABC transporters is characterized by the single ABC-trandomain and the overall amino acid sequence. Therefore, the completecoding sequence and not only the domains are compared. FIG. 17 shows thealignment of human, mouse, and Drosophila DevG22 proteins. DrosophilaDevG22 is 36% identical and 52% similar to human DevG22 (hWhite; ABC8,ABCG1, GenBank Accession Number XP_(—)009777). Drosophila DevG22 is 36%identical and 51% similar to mouse DevG22 (mWhite; GenBank AccessionNumber NP_(—)033723). Therefore, the vertebrate white transporter is theclosest homologue to Drosophila DevG22. Human and mouse White proteinsshow 95% identity and 96% similarity. Based upon homology, DevG22protein of the invention and each homologous protein or peptide mayshare at least some activity.

Example 9 Expression of the Polypeptides in Mammalian Tissues

For analyzing the expression of the polypeptides disclosed in thisinvention in mammalian tissues, several mouse strains (preferrably micestrains C57BI/6J, C57BI/6 ob/ob and C57BI/KS db/db which are standardmodel systems in obesity and diabetes research) were purchased fromHarlan Winkelmann (33178 Borchen, Germany) and maintained under constanttemperature (preferrably 22° C.), 40 percent humidity and a light/darkcycle of preferrably 14/10 hours. The mice were fed a standard chow (forexample, from ssniff Spezialitäten GmbH, order number ssniff M-ZV1126-000). Animals were sacrificed at an age of 6 to 8 weeks. Theanimal tissues were isolated according to standard procedures known tothose skilled in the art, snap frozen in liquid nitrogen and stored at−80° C. until needed.

For analyzing the role of the proteins disclosed in this invention inthe in vitro differentiation of different mammalian cell culture cellsfor the conversion of pre-adipocytes to adipocytes, mammalian fibroblast(3T3-L1) cells (e.g., Green & Kehinde, Cell 1: 113–116, 1974) wereobtained from the American Tissue Culture Collection (ATCC, Hanassas,Va., USA; ATCC-CL 173). 3T3-L1 cells were maintained as fibroblasts anddifferentiated into adipocytes as described in the prior art (e.g., Qiu.et al., J. Biol. Chem. 276:11988–95, 2001; Slieker et al., BBRC 251:225–9, 1998). At various time points of the differentiation procedure,beginning with day 0 (day of confluence) and day 2 (hormone addition;for example, dexamethason and 3-isobutyl-1-methylxanthin), up to 10 daysof differentiation, suitable aliquots of cells were taken every twodays. Alternatively, mammalian fibroblast 3T3-F442A cells (e.g., Green &Kehinde, Cell 7: 105–113, 1976) were obtained from the Harvard MedicalSchool, Department of Cell Biology (Boston, Mass., USA). 3T3-F442A cellswere maintained as fibroblasts and differentiated into adipocytes asdescribed previously (Djian, P. et al., J. Cell. Physiol., 124:554–556,1985). At various time points of the differentiation procedure,beginning with day 0 (day of confluence and hormone addition, forexample, Insulin), up to 10 days of differentiation, suitable aliquotsof cells were taken every two days. 3T3-F442A cells are differentiatingin vitro already in the confluent stage after hormone (insulin)addition.

RNA was isolated from mouse tissues or cell culture cells using TrizolReagent (for example, from Invitrogen, Karlsruhe, Germany) and furtherpurified with the RNeasy Kit (for example, from Qiagen, Germany) incombination with an DNase-treatment according to the instructions of themanufacturers and as known to those skilled in the art. Total RNA wasreverse transcribed (preferrably using Superscript II RNaseH ReverseTranscriptase, from Invitrogen, Karlsruhe, Germany) and subjected toTaqman analysis preferrably using the Taqman 2×PCR Master Mix (fromApplied Biosystems, Weiterstadt, Germany; the Mix contains according tothe Manufacturer for example AmpliTaq Gold DNA Polymerase, AmpErase UNG,dNTPs with dUTP, passive reference Rox and optimized buffer components)on a GeneAmp 5700 Sequence Detection System (from Applied Biosystems,Weiterstadt, Germany).

For the analysis of the expression of DevG20, taqman analysis wasperformed using the following primer/probe pair (see FIG. 6): MouseDevG20 (PDI) forward primer (SEQ ID NO:13): 5′-CAC GGG TGA CAA GGGCA-3′; mouse DevG20 (PDI) reverse primer (SEQ ID NO:14): 5′-CCC CTG TGCAAT AGT GTC CTC-3′; Taqman probe (SEQ ID NO:15): (5/6-FAM) TGC TGG CACTCA CCG AGA AGA GCT T (5/6-TAMRA).

As shown in FIG. 6A, real time PCR (Taqman) analysis of the expressionof DevG20 protein in mammalian (mouse) tissues revealed that DevG20(PDI) is rather ubiquitously expressed in various mouse tissues.However, a clear expression in WAT and BAT can also be demonstrated.DevG20 (PDI) shows an up-regulation of its expression in BAT, cortex andspleen of genetically obese ob/ob mice (FIG. 6B). In addition, itsexpression in kidney and bone marrow of fasted mice is alsoup-regulated. Even though no up-regulation of DevG20 (PDI) expression inWAT of ob/ob mice has been observed, we can clearly demonstrate atwo-fold up-regulation of its expression during the in vitrodifferentiation of 3T3-L1 cells from pre-adipocytes to mature adipocytes(FIG. 6C).

For the analysis of the expression of DevG4, taqman analysis wasperformed using the following primer/probe pair (see FIG. 12): MouseDevG4 (mrp4) forward primer (SEQ ID NO:16): 5′-CAA GTA GCG CCC ACCCC-3′; Mouse DevG4 (mrp4) reverse primer (SEQ ID NO:17): 5′-AGT TCA CATTGT CGA AGA CGA TGA-3′; Taqman probe (SEQ ID NO:18): (5/6-FAM) AGG CTGGCC CCA CGA GGG A (5/6-TAMRA).

Taqman analysis revealed that DevG4 (mrp4) is ubiquitously expressed invarious mouse tissues with highest levels of expression found in kidney(FIG. 12A). DevG4 (mrp4) shows a very prominent up-regulation of itsexpression in liver of genetically obese ob/ob mice (FIG. 12B). Inaddition, a significant up-regulation in kidney can also be observedunder these conditions. Under fasting conditions, DevG4 (mrp4)expression seems to show a global down-regulation of its expression,this is especially prominent in the BAT tissue of fasting mice. DevG4(mrp4) expression increases approximately 4-fold during the in vitrodifferentiation of 3T3-L1 cells from pre-adipocytes to mature adipocytes(FIG. 12C).

For the analysis of the expression of DevG22, taqman analysis wasperformed using the following primer/probe pair: Mouse DevG22 (white)forward primer (SEQ ID NO:19):5′-TCG TAT ACT GGA TGA CGT CCC A-3′; MouseDevG22 (white) reverse primer (SEQ ID NO:20): 5′-TGG TAC CCA GAG CAG CGAAC-3′; Taqman probe (SEQ ID NO:21): (5/6-FAM) CCG TCG GAC GCT GTG CGTTTT (5/6-TAMRA).

Taqman analysis revealed that DevG22 (white) is predominantly expressedin neuronal tissues. However, a clear expression in other tissues likeWAT or BAT has also been noted (FIGS. 18A and 18B). The expression ofDevG22 (white) in BAT and WAT is under metabolic control: In fastedmice, expression goes up in BAT. Contrary to this, expression isincreased in WAT and muscle in genetically obese ob/ob mice (FIG. 18B).This up-regulation in ob/ob mice correlates with the observed strongup-regulation of DevG22 (white) expression during the in vitrodifferentiation of 3T3-L1 cells (FIG. 18C).

All publications and patents mentioned in the above specification areherein incorporated by reference.

Various modifications and variations of the described method and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

LITERATURE

-   Klucken J, Büchler C, Orsó E, Kaminski W E, Porsch-Özcürümez M,    Liebisch G, Kapinsky M, Diederich W, Drobnik W, Dean M, Allikmets R,    Schmitz G.: ABCG1 (ABC8), the human homolog of the Drosophila white    gene, is a regulator of macrophage cholesterol and phospholipid    transport. Proc Natl Acad Sci USA. 2000 January;97(2):817–22.-   Orsó E, Broccardo C, Kaminski WE, Böttcher A, Liebisch G, Drobnik W,    Götz A, Chambenoit O, Diederich W, Langmann T, Spruss T, Luciani    M-F, Rothe G, Lackner K J, Chimini G, Schmitz G.: Transport of    lipids from Golgi to plasma membrane is defective in Tangier disease    patients and Abc1-deficient mice. Nat Genet. 2000 February;24:192–6.-   Venkateswaran A, Repa J J, Lobaccaro J-M A, Bronson A, Mangelsdorf D    J, Edwards P A.: Human White/murine ABC8 mRNA levels are highly    induced in lipid-loaded macrophages. J Biol Chem. 2000    May;275(19):14700–7. Nakamura M, Ueno S, Sano A, Tanabe H.:    Polymorphisms of the human homologue of the Drosophila white gene    are associated with mood and panic disorders. Mol Psychiatry. 1999    March;4(2):155–62.-   Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, Diederich W,    Drobnik W, Barlage S, Buchler C, Porsch-Ozcurumez M, Kaminski W E,    Hahmann H W, Oette K, Rothe G, Aslanidis C, Lackner K J, Schmitz G.:    The gene encoding ATP-binding cassette transporter 1 is mutated in    Tangier disease. Nat Genet. 1999 August;22(4):347–51.-   Brooks-Wilson A, Marcil M, Clee S M, Zhang L H, Roomp K, van Dam M,    Yu L, Brewer C, Collins J A, Molhuizen H O, Loubser O, Ouelette BF,    Fichter K, Ashbourne-Excoffon K J, Sensen C W, Scherer S, Mott S,    Denis M, Martindale D, Frohlich J, Morgan K, Koop B, Pimstone S,    Kastelein J J, Hayden M R, et al.: Mutations in ABC1 in Tangier    disease and familial high-density lipoprotein deficiency. Nat Genet.    1999 August;22(4):336–45.-   Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette J C, Deleuze J    F, Brewer H B, Duverger N, Denefle P, Assmann G.: Tangier disease is    caused by mutations in the gene encoding ATP-binding cassette    transporter 1. Nat Genet. 1999 August;22(4):352–5.-   Schuetz J D, Connelly M C, Sun D, Paibir S D, Flynn P M, Srinivas R    V, Kumar A, Fridland A.: MRP4. A previously unidentified factor in    resistance to nucleoside-based antiviral drugs. Nat Med. 1999    September;5(9):1048–51.-   Wada M, Toh S, Taniguchi K, Nakamura T, Uchiumi T, Kohno K, Yoshida    I, Kimura A, Sakisaka S, Adachi Y, Kuwano M.: Mutations in the    canilicular organic anion transporter (cMOAT) gene, a novel ABC    transporter, in patients with hyperbilirubinemia II/Dubin-Johnson    syndrome. Hum Mol Genet. 1998 February;7(2):203–7.-   Allikmets R, Singh N, Sun H, Shroyer NF, Hutchinson A, Chidambaram    A, Gerrard B, Baird L, Stauffer D, Peiffer A, Rattner A, Smallwood    P, Li Y, Anderson K L, Lewis R A, Nathans J, Leppert M, Dean M,    Lupski J R.: A photoreceptor cell-specific ATP-binding transporter    gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat    Genet. 1997 March; 15 (3):236–46.

1. A composition comprising an isolated ABC transporter DevG22polypeptide encoded by a polynucleotide sequence as defined in SEQ IDNO:5, and an acceptable carrier, wherein said ABC transporter DevG22polypeptide regulates energy homeostasis and metabolism oftriglycerides.
 2. The composition of claim 1, wherein the ABCtransporter DevG22 polypepride comprises an amino acid sequence setforth in SEQ ID NO:6.
 3. The composition of claim 1, wherein the ABCtransporter DevG22 polypeptide is a recombinant polypeptide.
 4. Thecomposition of claim 1, wherein the ABC transporter DevG22 polypeptideis a recombinant fusion polypeptide.
 5. The composition of claim 1,wherein the composition is a diagnostic composition.